| PurposeEndometriosis is defined as the implantation of endometrium- like glandular and stromal cells outside their normal location in the uterus. Overall, studies estimate that endometriosis may affect around 10-15% of women of reproductive age, thus making this a common condition. Although endometriosis is a benign disorder, recent studies of endometriosis suggest endometriosis could be viewed as a neoplastic process. Knowledge of the mechanism controlling endometriotic cell growth and differentiation is also limited.Currently, the favored paradigm for the establishment of endometriosis is the "reigning endometrial cell determinism theory" that postulates that the mutation of gene and protein in viable endometrial cells may an essential factor in the development of endometriosis, and affect the process of endometrial cells being transported to the peritoneal cavity by retrograde menstruation, adhering to the peritoneal wall, proliferating, and forming lesions. Protein genomic studies from our group suggest that annexin I was may be associated with the pathogenesis of endometriosis.Annexin I is the first characterized member of the annexin family of proteins able to bind to cellular membranes or cytoskeleton in a calcium-dependent manner. Annexin I is one of structurally related phospholipids-biding proteins that had been implicated in diverse cellular roles, including anti-inflammatory, signaling transduction, cell differentiation, membrane aggregation, inhibited the activity of cytosolic phospholipase A2, calcium channels, and interaction with cytoskeleton. Originally described as a (PLA2)-inhibitory protein, Annexin I may specifically target cytosolic PLA2 to inhibit the expression and/or activity of inducible cyclooxygenase (COX-2) and prostaglandins (PGE2). Recent data have shown that Annexin I can interacted with many molecules, such as S100 families, F-action, epithelial growth factor receptor, cytosolic PLA2 andα4β1 integrin. The N-terminal domain of annexin I contains phosphorylation sites for several tyrosine kinases including the epidermal growth factor (EGF) receptor tyrosine kinase, which phosphorylates tyrosine 21. A link between annexin I and EGFR signaling can specifically target mitogen-activated protein kinases signaling to participate in cellular function.To date, there are various data on annexin I expression levels in human cancers. Annexin I overexpression has been observed in gastric and breast cancer, but loss or dramatic reduction of annexin I protein expression in squamous cell carcinoma of the esophagus and prostate cancer has been reported recently. The expression of annexin I was increased in atypical endometrial hyperplasia and decreased in endometrial carcinoma. Many studies of endometriosis suggest endometriosis could be viewed as a neoplastic process and give us a clue to investigate the alteration of endometrial cell function in endometriosis from the cancer cell biology.In this study, we first collected some endometrial tissue in endometriosis from clinical patient and address annexin I relevant to the clinical pathophysiology in endometriosis. Secondly, we isolated and culture the reigning endometrial cell an invtro. Based on the invtro model, we further to study the function of annexin I in endometriosis.Methods一,Collect samples and detect the expression of annexin I1 Subjects: Women with ovulatory menstrual cycles, who had not received hormones or GnRH agonist therapy for at least 3 months before surgery, were enrolled in the study. Subjects undergoing elective surgery for leiomyomata uteri requiring hysterectomy were recruited for normal endometrial biopsies. Subjects with an echogenic adnexal mass documented on ultrasonography were recruited for the possible retrieval of endometrioma biopsies at surgery. All samples were histologically confirmed.2 The expression of annexinI was detected by S-P immunohistochemisrty.3 The histological distribution of annexin I was detected by immunofluroescence4 The level of annexin I mRNA was detected by real time PCR.5 The level of annexin I protein was detected by western blot.二,Cell isolation, purification and identification the reigning endometrial cell an invtro1 Collection the endometrial tissue2 The tissue was dissected free and digested with collagenase, and separated using serial filtration.3 Cell morphology changes of living cell photographed under phase-contrast microscope.4 Identification of endometrial cell by immunohistochemistry.5 Identification of endometrial cell by immunofluroescence.6 The expression of annexin I in endometrial cell was detected by immunohistochemistry.7 The expression of annexin I in endometrial cell was detected by immunofluroescence.三,The molecular mechanism of annexin I in endometriosis1 Endometrial cell isolation and purification2 Culturing the endometrial cell with medium containing EGF.3 Cell morphology changes and adhesion of living cell photographed under phase-contrast microscope.4 The levelsα4β1 integrin and annexin I in the endometrial cell treated with EGF are detected by western blot.5 The colocalization ofα4β1 integrin and annexin I in the endometrial cell treated with EGF are detected by immunofluroescence.6 The binding ofα4β1integrin and annexin I in the endometrial cell are detected by coimmunoprecipitation.7 The binding ofα4β1integrin and annexin I in the endometrial cell treated with EGF are detected by coimmunoprecipitation.8 The level of phospholated annexin I in the endometrial cell treated with EGF are detected by immunoprecipitation.Results1. Endometrial tissue samples were collected from 35 reproductive-age women with endometriosis who were undergoing surgery. 10 endometrial tissue samples were collected from woman with leiomyomata uteri requiring hysterectomy as control.2. The result of real time PCR showed that the level of annexin I mRNA in endometriosis was lower than in control. Western blot results showed the similar results.3. Immunohistochemistry and immunofluroescence results showed that the distribution of annexin I in endometriosis was agree with the control, which was localized within the endometrial glad cells membrane. Semi-quantitative analysis of immunohistochemistry was also consistent with the above results.4. We have successfully established an in vitro model to investigate phenotypic similarities and differences between normal endometrial and endometriosis cells. Highly purified cultures of epithelial and stromal cells were isolated from normal endometrium and endometriosis implants. Morphological features as well as immunocytochemical markers confirm these isolates as epithelial and stromal cells.5. In vitro system, annexin I was mainly localized in the epithelial cell membrane and colocalized with cytokeratin, the immunocytochemical marker of epithelial cell.6. EGF improved the adhesion of glad epithelail cell in endometriosis compared with the control.7. Compared with the control, the level ofα4β1integrin in glad epithelail cell from endometriosis was no significant alteration after being treated with EGF. The level of annexin I was not only reduced in glad epithelail cell from endometriosis, but also lower after being treated with EGF.8. In normal glad epithelail cell, annexin I not only colocalized withα4β1integrin but also bind toα4β1integrin. These results suggest that the interaction ofα4β1integrin and annexin I may be associated with cell adhesion.9. EGF has no effect on the level ofα4β1integrin bind to annexin I in normal glad epithelail cell, but reduced the amount ofα4β1integrin bind to annexin I in glad epithelail cell from endometriosis.10. EGF significantly improved the level of phospholated annexin I in glad epithelail cell from endometriosis.Conclusions1. Clinical data showed the expression of annexin I was significantly decreased in endometrial tissue from endometriosis, which was consistent with "reigning endometrial cell determinism theory".2. According to modified methods, we have successfully established an in vitro model to investigate phenotypic similarities and differences between normal endometrial and endometriosis cells.3. In vitro system, we found that the expression of annexin I in cultured glad epithelia cells was decreased, which may be affect the function of endometrial cell adhesion by reducing the number ofα4β1integrin bind to annexin I. |
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